Methods and compositions for modulating axonal outgrowth of central nervous system neurons

ABSTRACT

Methods and compositions for modulating the axonal outgrowth of central nervous system neurons are provided. Methods for stimulating the axonal outgrowth of central nervous system neurons following an injury (e.g., stroke, Traumatic Brain Injury, cerebral aneurism, spinal cord injury and the like) and methods for inhibiting the axonal outgrowth of central nervous system neurons in conditions such as epilepsy, e.g., posttraumatic epilepsy, and neuropathic pain syndrome, are also provided. These methods generally involve contacting the central nervous system neurons with a compound that modulates the activity of N-kinase, or analog thereof. The methods and compositions are particularly useful for modulating the axonal outgrowth of mammalian central nervous system neurons, such as mammalian cortical neurons or retinal ganglion cells. Pharmaceutical and packaged formulations that include the compounds of the invention that modulate the activity of N-kinase are also provided.

BACKGROUND OF THE INVENTION

Past early childhood, injury to the central nervous system (CNS) resultsin functional impairments that are largely irreversible. Within thebrain or spinal cord, damage resulting from stroke, trauma, or othercauses can result in life-long losses in cognitive, sensory and motorfunctions, and even maintenance of vital functions. Nerve cells that arelost are not replaced, and those that are spared are generally unable tore-grow severed connections, although a limited amount of local synapticreorganization can occur close to the site of injury. Functions that arelost are currently untreatable.

Regenerative failure in the CNS has been attributed to a number offactors, which include the presence of inhibitory molecules on thesurface of glial cells that suppress axonal growth; absence ofappropriate substrate molecules such as laminin to foster growth and anabsence of the appropriate trophic factors needed to activate programsof gene expression required for cell survival and differentiation.

By contrast, within the peripheral nervous system (PNS), injured nervefibers can re-grow over long distances, with eventual excellent recoveryof function. Within the past 15 years, neuroscientists have come torealize that this is not a consequence of intrinsic differences betweenthe nerve cells of the peripheral and central nervous system;remarkably, neurons of the CNS will extend their axons over greatdistances if given the opportunity to grow through a grafted segment ofPNS (e.g., sciatic nerve). Therefore, neurons of the CNS retain acapacity to grow if given the right signals from the extracellularenvironment. Factors which contribute to the differing growth potentialsof the CNS and PNS include certain growth-inhibiting molecules on thesurface of the oligodendrocytes that surround nerve fibers in the CNS,but which are less abundant in the comparable cell population of the PNS(Schwann cells); molecules of the basal lamina and other surfaces thatfoster growth in the PNS but which are absent in the CNS (e.g.,laminin); and trophic factors, soluble polypeptides which activateprograms of gene expression that underlie cell survival anddifferentiation. Although such trophic factors are regarded as essentialfor maintaining the viability and differentiation of nerve cells, theparticular ones that are responsible for inducing axonal regeneration inthe CNS remain uncertain.

Moreover, the intracellular molecule(s) that mediates axonal outgrowthof normal neuronal cells (e.g., upon stimulation with extracellularfactors or upstream secondary messengers) has not been elucidated. Onereport has described the partial isolation of a kinase, referred to as“protein kinase N”, from rat pheochromocytoma PC12 cells that isactivated by NGF treatment of the PC12 cells and sensitive to purineregulation (C. Volonte, et al., (1989) J. Cell Biol. 109, 2395-403).However, as PC12 cells are a rat phaeochromocytoma cell line from theadrenal medulla, with many different properties than normal CNS neurons,these cells present a limited model for the processes by which growth ofnormal CNS neurons is stimulated and the results obtained in PC12 cellsmay not be predictive of molecules involved in normal CNS neuron growth.Furthermore, this protein kinase N was only partially purified andremains to be molecularly characterized.

In view of the lack of understanding of the molecules involved inmediating axonal outgrowth, effective treatments for CNS injuries havenot been developed. Accordingly, elucidation and molecularcharacterization of such molecules is still necessary, and methods andcompositions for modulating the outgrowth of normal CNS neurons bymodulating the activity of such molecules are still needed.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for modulatingthe axonal outgrowth of central nervous system neurons, in particularnormal mammalian central nervous system neurons. The invention is based,at least in part, on the isolation of a highly purified form of theN-kinase polypeptide from normal mammalian neuronal tissue, molecularcharacterization of its chemical structure (including amino acidsequence), demonstration of its sensitivity to purine regulation and thediscovery that this kinase plays an active role in the axonal outgrowthof CNS neurons, including mammalian CNS neurons, such as retinalganglion neurons. Identification of N-kinase as a critical intracellularmediator of axonal outgrowth, and chemical characterization of itsstructure, now provides for the ability to modulate axonal outgrowth bymodulating N-kinase activity. Furthermore, this purification andcharacterization of N-kinase now allows for its use in screening assaysto identify additional modulators of axonal outgrowth.

Accordingly, in one aspect, the present invention is directed to amethod for treating a subject (e.g., a mammal, such as a human)suffering or prone to suffering from a condition characterized byaberrant, e.g., inadequate or insufficient, axonal outgrowth of centralnervous system neurons (e.g., stroke, CNS trauma, or a neurodegeneratedisease), by administering to the subject a compound that modulates theactivity of N-kinase, thereby treating the subject suffering or prone tosuffering from a condition characterized by aberrant axonal outgrowth ofcentral nervous system neurons.

The methods of the invention for modulating, e.g., stimulating, theaxonal outgrowth of central nervous system neurons can be used followingdamage or other injury to the CNS neurons (e.g., stroke, Traumatic BrainInjury, cerebral aneurism, spinal cord injury and the like). The methodsof the invention for modulating, e.g., inhibiting, the axonal outgrowthof CNS neurons can be used in neuroproliferative disorders whereaberrant axonal outgrowth may occur, such as epilepsy (e.g.,post-traumatic epilepsy) and neuropathic pain syndrome.

In one aspect, the compound that modulates the activity of N-kinase isadministered to a subject in accordance with the present invention byintroduction into the central nervous system of the subject, for exampleinto the cerebrospinal fluid of the subject. In certain aspects of theinvention, the compound that modulates the activity of N-kinase isintroduced intrathecally, for example into a cerebral ventricle, thelumbar area, or the cisterna magna. In a preferred embodiment, themethod of the invention modulates outgrowth of damaged cortical neurons.In yet another preferred embodiment, the method of the inventionmodulates outgrowth of damaged retinal ganglion cells. In suchcircumstances, the compound that modulates the activity of N-kinase canbe administered locally to cortical neurons or retinal ganglion cells tomodulate axonal outgrowth.

In yet another aspect of the invention, the compound that modulates theactivity of N-kinase is administered in a pharmaceutically acceptableformulation. The pharmaceutically acceptable formulation can be adispersion system, for example a lipid-based formulation, a liposomeformulation, or a multivesicular liposome formulation. Thepharmaceutically acceptable formulation can also comprise a polymericmatrix, selected, for example, from synthetic polymers such aspolyesters (PLA, PLGA), polyethylene glycol, poloxomers, polyanhydrides,and pluronics or selected from naturally derived polymers, such asalbumin, alginate, cellulose derivatives, collagen, fibrin, gelatin, andpolysaccharides.

In a preferred embodiment, the pharmaceutically acceptable formulationprovides sustained delivery, e.g., “slow release” of the compound thatmodulates the activity of N-kinase to a subject for at least one week,more preferably at least one month, after the pharmaceuticallyacceptable formulation is administered to the subject. Preferredapproaches for achieving sustained delivery of a formulation of theinvention include the use of a slow release polymeric capsules or aninfusion pump that includes the formulation.

In one embodiment of the invention, the compound that modulates theactivity of N-kinase is a small molecule, the N-kinase polypeptide orfragment thereof, an anti-N-kinase antibody, an antisense N-kinasenucleic acid molecule, a ribozyme, or the N-kinase gene or fragmentthereof.

In another aspect, the invention features a method for modulating, e.g.,stimulating or inhibiting, axonal outgrowth of a central nervous systemneuron (such as a mammalian central nervous system neuron) by contactingthe central nervous system neuron with a compound that modulates theactivity of N-kinase, thereby modulating axonal outgrowth of the centralnervous system neuron.

In yet another aspect, the invention features a method for modulatingthe axonal outgrowth of a central nervous system neuron in a subject, byadministering to the subject a compound that modulates the activity ofN-kinase, such that axonal outgrowth in the subject is modulated.

In a further aspect, the invention features a method for identifying acompound that modulates axonal outgrowth of a central nervous systemneuron by contacting N-kinase with a test compound and determining theability of the test compound to modulate the activity of N-kinase,thereby identifying a compound that modulates axonal outgrowth of acentral nervous system neuron. In a preferred embodiment, the ability ofthe test compound to modulate the activity of N-kinase is determined byassessing the ability of the test compound to modulate N-kinasedependent phosphorylation of a substrate, e.g., a histone HF-1 protein.

In one embodiment, the N-kinase used in the methods of the invention isa human N-kinase, such as a recombinantly produced human N-kinase. Inanother embodiment, the N-kinase used in the methods of the invention isa bovine N-kinase, such as an N-kinase which is purified from a bovinesource, e.g., neonatal bovine brain tissue.

In another embodiment, the screening method of the invention furtherincludes determining the ability of the test compound to modulate axonaloutgrowth of a central nervous system neuron.

In another aspect, the invention features a method for identifying acompound that modulates axonal outgrowth of a central nervous systemneuron, comprising contacting N-kinase with a test compound, an N-kinasesubstrate (e.g., a histone HF-1 protein), radioactive ATP (e.g., [γ-³²P]ATP), and Mn⁺²; and determining the ability of the test compound tomodulate N-kinase dependent phosphorylation of the substrate, therebyidentifying a compound that modulates axonal outgrowth of a centralnervous system neuron. In a preferred embodiment, the method of theinvention further includes determining the ability of the test compoundto modulate axonal outgrowth of a central nervous system neuron.

In another aspect, the invention features a compound that modulatesaxonal outgrowth of a central nervous system neuron identified by any ofthe foregoing methods.

In yet another aspect, the invention features an isolated N-kinasepolypeptide of the type that: (a) is present in neonatal brain tissue(e.g., neonatal human, bovine, rat, or mouse brain tissue); (b) isinhibited by 6-thioguanine; (c) is activated by Mn⁺² but not by Mg⁺² orCa⁺²; (d) has a molecular weight of approximately 49 kDa; and (e) iseluted from a Cibacron Blue column at a NaCl concentration of 1.5-1.75M.

In a further aspect, the invention features an antibody, e.g., anintracellular antibody, which is specifically reactive with an epitopeof the N-kinase polypeptide. In a preferred embodiment, the antibody isreactive with an epitope which includes the ATP binding domain of theN-kinase.

In another aspect, the invention features a fragment of the N-kinasepolypeptide, for example, a fragment that includes at least 15, 20, 25,30, 40, 50, 100, 150, or 200 contiguous amino acids of the N-kinasepolypeptide. In a preferred embodiment, the fragment of the N-kinasepolypeptide is able to elicit an immune response.

Pharmaceutical compositions, and packaged formulations, comprising acomposition of the invention (e.g., compound that modulates the activityof N-kinase) and a pharmaceutically acceptable carrier are also providedby the invention.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C are SDS-PAGE gels depicting the purification of the N kinasepolypeptide. The N-kinase band at each stage of the purification isindicated by an asterisk. FIG. 1A depicts a prominent 49 kDa band whichbinds strongly to a Cibacron Blue column and requires 1.5-1.75 M NaCl tobe eluted. FIG. 1B depicts the protein fractions obtained from theseparation on a C4 hydrophobic-interaction column. Fractions 24-26contain the N-kinase polypeptide. FIG. 1C depicts the final stage ofpurification which was accomplished by SDS-PAGE. The band indicated bythe asterisk coincided in its migration position with N-kinase activity,as visualized in a parallel gel assayed with the in-gel kinase method.

FIG. 2 depicts the amino acid (SEQ ID NO:1) sequence of the humanN-kinase. Direct matches between the purified protein and the publishedsequence are shown in blue. K65 (bold type) lies in the ATP-bindingregion of the kinase domain.

DETAILED DESCRIPTION

The present invention provides methods and compositions for modulatingthe axonal outgrowth of central nervous system neurons, in particularmammalian central nervous system neurons. The invention is based, atleast in part, on the isolation of a highly purified form of theN-kinase polypeptide from a mammalian source and the discovery that thiskinase plays an active role in the axonal outgrowth of CNS neurons,including mammalian CNS neurons, such as retinal ganglion neurons, orcortical pyramidal cells.

Accordingly, the present invention is directed to a method for treatinga subject (e.g., a mammal, such as a human) suffering or prone tosuffering from a condition characterized by aberrant axonal outgrowth ofcentral nervous system neurons, (e.g., a condition characterized by afailure of injured central nervous system neurons to regrow theirconnections) by administering to the subject a compound that modulatesthe activity of N-kinase, thereby treating the subject suffering orprone to suffering from a condition characterized by aberrant axonaloutgrowth of central nervous system neurons.

The methods of the invention for modulating, e.g., stimulating, theaxonal outgrowth of central nervous system neurons can be used followingdamage or other injury to the CNS neurons (e.g., stroke, Traumatic BrainInjury, cerebral aneurism, spinal cord injury and the like). The methodsof the invention for modulating, e.g., inhibiting, the axonal outgrowthof CNS neurons can be used in neuroproliferative disorders whereaberrant axonal outgrowth may occur, such as epilepsy (e.g.,post-traumatic epilepsy) and neuropathic pain syndrome.

As used herein, the term “N-kinase” includes all forms of N-kinaseincluding but not limited to human N-kinase, bovine N-kinase, murineN-kinase, rat N-kinase, and porcine N-kinase. The amino acid andnucleotide sequences of the human N-kinase are described in Zhou T-H. etal. (2000) J. Biol. Chem. 275(4):2513-2519 and in GenBank AccessionNumber AF083420, the contents of which are incorporated herein byreference. The amino acid sequence of the human N-kinase is shown inFIG. 2 and in SEQ ID NO:1. In a preferred embodiment, the term“N-kinase” includes the isoform of N-kinase that is inhibited by6-thioguanine, that is activated by Mn⁺² but not by Mg⁺² or Ca⁺², andthat has a molecular weight of approximately 49 kDa.

As used herein, the language “a compound that modulates the activity ofN-kinase” includes any compound which has the ability to modulate, e.g.,stimulate or inhibit, the activity of N-kinase as determined by, forexample, the assays described herein. Such compounds are able tomodulate one or more of the following: (a) the ability of N-kinase tophosphorylate a substrate, e.g., a histone HF-1 protein; (b) the abilityof N-kinase to interact with, e.g., bind to, a non-N-kinase molecule,such as a downstream molecule in the axonal outgrowth signaling pathway;(c) the ability of N-kinase to bind ATP or Mn⁺²; or (d) the ability ofN-kinase to modulate the axonal outgrowth of central nervous systemneurons.

In a preferred embodiment, the compound that modulates the activity ofN-kinase acts downstream of AF-1 or other growth factors in the axonaloutgrowth signaling pathway. The ability of the compound to actdownstream of AF-1 or other growth factors in the axonal outgrowthsignaling pathway may be determined using one of the assays describedherein. For example, once a compound has been determined to be capableof stimulating the activity of N-kinase (e.g., stimulating the N-kinasedependent phosphorylation of a substrate), N-kinase may be contactedboth with this compound and with 6-thioguanine. The inability of6-thioguanine to inhibit the stimulatory effect of the compound wouldindicate that the compound is acting downstream of 6-thioguanine in theaxonal outgrowth signaling pathway, whereas the ability of 6-thioguanineto inhibit the stimulatory effect of the compound would indicate thatthe compound is acting upstream (or at the same point) in the signalingpathway. Alternatively, once a compound has been determined to becapable of inhibiting the activity of N-kinase (e.g., inhibiting theN-kinase dependent phosphorylation of a substrate), N-kinase may becontacted both with this compound and with inosine. The inability ofinosine to counteract the inhibitory effect of the compound wouldindicate that the compound is acting downstream of inosine in the axonaloutgrowth signaling pathway, whereas the ability of inosine tocounteract the inhibitory effect of the compound would indicate that thecompound is acting upstream (or at the same point) in the signalingpathway.

In certain embodiments of the invention, the compound that modulates theactivity of N-kinase can be any compound with the proviso that it is nota purine base (e.g., guanine, inosine, adenosine, and xanthine), such asa purine base linked to sugars, such as ribose, deoxyribose, and analogsand derivatives thereof. In certain other embodiments of the invention,the compound that modulates the activity of N-kinase can be any compoundwith the proviso that it is not a purine base analog or derivativethereof.

Examples of compounds that modulate the activity of N-kinase includesmall molecules, the N-kinase polypeptide or fragments thereof, ananti-N-kinase antibody, an antisense N-kinase nucleic acid molecule, aribozyme, or the N-kinase gene or fragments thereof.

As used herein, the term “small molecule” includes, but is not limitedto, peptides, peptidomimetics, amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e.,. including heteroorganicand organometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds.

As used herein, the language “modulating the axonal outgrowth of centralnervous system neurons” is intended to include the capacity to stimulateor inhibit axonal outgrowth of central nervous system neurons to variouslevels, e.g., to levels which allow for the treatment of a targeted CNScondition.

As used herein, the term “outgrowth” (i.e., axonal outgrowth) refers tothe process by which axons grow out of a CNS neuron. The outgrowth canresult in a totally new axon or the repair of a partially damaged axon.Outgrowth is typically evidenced by extension of an axonal process of atleast 5 cell diameters in length. Moreover, axonal outgrowth can beevidenced by GAP-43 expression (which can be detected by, for example,immunostaining).

As used herein, the term “CNS neurons” is intended to include theneurons of the brain and the spinal cord which are unresponsive to nervegrowth factor (NGF). The term is not intended to include support orprotection cells such as astrocytes, oligodentrocytes, microglia,ependyma and the like, nor is it intended to include peripheral nervoussystem (e.g., somatic, autonomic, sympathetic or parasympathetic nervoussystem) neurons. Preferred CNS neurons are mammalian neurons, morepreferably human neurons.

As used herein, the language “contacting” is intended to include both invivo or in vitro methods of bringing a compound that modulates theactivity of N-kinase into proximity with a CNS neuron, such that thecompound that modulates the activity of N-kinase can modulate theoutgrowth of axonal processes from the CNS neuron.

As used herein, the term “subject” is intended to include animalssusceptible to conditions characterized by aberrant, e.g., insufficient,axonal outgrowth of central nervous system neurons, preferably mammals,most preferably humans. In a preferred embodiment, the subject is aprimate. In an even more preferred embodiment, the primate is a human.Other examples of subjects include dogs, cats, goats, and cows.

As used herein, the term “condition characterized by aberrant axonaloutgrowth of central nervous system neurons” is intended to include adisease, disorder, or condition which is caused or characterized by anaberrant, e.g., increased, insufficient, inadequate, or decreased,axonal outgrowth of central nervous system neurons. Such conditionsdirectly or indirectly affect the normal functioning of the centralnervous system (CNS). A condition characterized by aberrant, e.g.,insufficient, axonal outgrowth of central nervous system neuronsincludes, but is not limited to, an injury to the optic nerve, e.g.,affecting retinal ganglion cells; traumatic brain injury; stroke;cerebral aneurism; spinal cord injury, including monoplegia, diplegia,paraplegia, hemiplegia and quadriplegia; neuroproliferative disorders,e.g., Alzheimer's disease, dementias related to Alzheimer's disease(such as Pick's disease), Parkinson's and other Lewy diffuse bodydiseases, multiple sclerosis, amyotrophic lateral sclerosis, progressivesupranuclear palsy, epilepsy, Jakob-Creutzfieldt disease, or AIDSrelated dementias; epilepsy, e.g., posttraumatic brain injury; orneuropathic pain syndrome.

As used herein, the term “stroke” is art recognized and is intended toinclude sudden diminution or loss of consciousness, sensation, andvoluntary motion caused by rapture or obstruction (e.g., by a bloodclot) of an artery of the brain.

As used herein, the term “Traumatic Brain Injury” is art recognized andis intended to include the condition in which, a traumatic blow to thehead causes damage to the brain or connecting spinal cord, often withoutpenetrating the skull. Usually, the initial trauma can result inexpanding hematoma, subarachnoid hemorrhage, cerebral edema, raisedintracranial pressure (ICP), and cerebral hypoxia, which can, in turn,lead to severe secondary events due to low cerebral blood flow (CBF).

In another aspect, the invention features a method for modulating, e.g.,stimulating or inhibiting, axonal outgrowth of a central nervous systemneuron (such as a mammalian central nervous system neuron) by contactingthe central nervous system neuron with a compound that modulates theactivity of N-kinase, thereby modulating axonal outgrowth of the centralnervous system neuron.

In yet another aspect, the invention features a method for modulatingthe axonal outgrowth of a central nervous system neuron in a subject, byadministering to the subject a compound that modulates the activity ofN-kinase, such that axonal outgrowth in the subject is modulated.

In yet another aspect, the invention features an isolated N-kinasepolypeptide of the type that: (a) is present in neonatal brain tissue(e.g., neonatal human, bovine, rat, or mouse brain tissue); (b) isinhibited by 6-thioguanine; (c) is activated by Mn⁺² but not by Mg⁺² orCa⁺²; (d) has a molecular weight of approximately 49 kDa; and (e) iseluted from a Cibacron Blue column at a NaCl concentration of 1.5-1.75M. As used herein, an “isolated” N-kinase polypeptide is substantiallyfree (i.e., greater than 95% free) of cellular material or othercontaminating proteins from the cell or tissue source from which theN-kinase protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations ofN-kinase in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. In oneembodiment, the language “substantially free of cellular material”includes preparations of N-kinase protein having less than about 20% (bydry weight) of non-N-kinase protein (i.e., contaminating protein), morepreferably less than about 10% of non-N-kinase protein, still morepreferably less than about 5% of non-N-kinase protein, and mostpreferably less than about 3% non-N-kinase protein. When the N-kinaseprotein or biologically active portion thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, more preferablyless than about 10%, and most preferably less than about 5% of thevolume of the protein preparation.

Various aspects of the invention are described in further detail in thefollowing subsections:

Method for Identifying a Compound that Modulates Axonal Outgrowth of aCentral Nervous System Neuron

In one aspect, the invention features a method for identifying acompound that modulates axonal outgrowth of a central nervous systemneuron by contacting N-kinase with a test compound and determining theability of the test compound to modulate the activity of N-kinase,thereby identifying a compound that modulates axonal outgrowth of acentral nervous system neuron.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the ‘one-bead one-compound’ library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, non-peptide oligomer orsmall molecule libraries of compounds (Lam, K. S. (1997) Anticancer DrugDes. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses an N-kinase protein or biologically active portion thereof iscontacted with a test compound and the ability of the test compound tomodulate N-kinase activity is determined. Determining the ability of thetest compound to modulate N-kinase activity can be accomplished bymonitoring, for example, the production of one or more specificmetabolites in a cell which expresses N-kinase (see, e.g., Saada et al.(2000) Biochem Biophys. Res. Commun. 269: 382-386). The cell, forexample, can be of mammalian origin, e.g., a neuronal cell.

Determining the ability of the test compound to modulate N-kinaseactivity can further be accomplished by, for example, determining theability of N-kinase to phosphorylate a substrate. The ability ofN-kinase to phosphorylate a substrate (e.g., a histone HF-1 protein) canbe determined by, for example, an in vitro kinase assay. Briefly, theN-kinase can be incubated with the substrate and radioactive ATP, e.g.,[γ-³²P] ATP, in a buffer containing MnCl₂, e.g., 5 mM MnCl₂. Followingthe incubation, the substrate can be immunoprecipitated or precipitatedwith TCA or collected on a filter (if no other kinases are present) andseparated by SDS-polyacrylamide gel electrophoresis under reducingconditions, transferred to a membrane, e.g., a PVDF membrane, andautoradiographed. The appearance of detectable bands on theautoradiograph indicates that the substrate has been phosphorylated.Alternatively, the in-gel assays described in Example 1 may be used todetermine the ability of N-kinase to phosphorylate a substrate.Phosphoaminoacid analysis of the phosphorylated substrate can also beperformed in order to determine which residues on the protein arephosphorylated. Briefly, the radiophosphorylated protein band can beexcised from the SDS gel and subjected to partial acid hydrolysis. Theproducts can then be separated by one-dimensional electrophoresis andanalyzed on, for example, a phosphoimager and compared toninhydrin-stained phosphoaminoacid standards.

The ability of the test compound to modulate N-kinase binding to anon-N-kinase molecule, such as a downstream molecule in the axonaloutgrowth signaling pathway, can also be determined. Determining theability of the test compound to modulate N-kinase binding to anon-N-kinase molecule can be accomplished, for example, by coupling thenon-N-kinase molecule with a radioisotope or enzymatic label such thatbinding of the non-N-kinase molecule to N-kinase can be determined bydetecting the labeled non-N-kinase molecule in a complex.

It is also within the scope of this invention to determine the abilityof a test compound to interact with, e.g., bind to, N-kinase orbiologically active portions thereof. Preferred biologically activeportions of the N-kinase proteins to be used in assays of the presentinvention include fragments which participate in interactions withnon-N-kinase molecules, e.g., fragments with high surface probabilityscores. Determining the ability of the test compound to bind N-kinasecan be accomplished, for example, by coupling the compound with aradioisotope or enzymatic label such that binding of the compound toN-kinase can be determined by detecting the labeled N-kinase compound ina complex. For example, test compounds can be labeled with ¹²⁵I, ³⁵S,¹⁴C, or ³H, either directly or indirectly, and the radioisotope detectedby direct counting of radioemmission or by scintillation counting.Alternatively, test compounds can be enzymatically labeled with, forexample, horseradish peroxidase, alkaline phosphatase, or luciferase,and the enzymatic label detected by determination of conversion of anappropriate substrate to product.

Determining the ability of a test compound to interact with N-kinase mayalso be accomplished without the labeling of any of the interactants.For example, a microphysiometer can be used to detect the interaction ofa compound with N-kinase without the labeling of either the compound orthe N-kinase. McConnell, H. M. et al. (1992) Science 257:1906-1912. Asused herein, a “microphysiometer” (e.g., Cytosensor) is an analyticalinstrument that measures the rate at which a cell acidifies itsenvironment using a light-addressable potentiometric sensor (LAPS).Changes in this acidification rate can be used as an indicator of theinteraction between a compound and N-kinase.

Determining the ability of the test compound to bind to N-kinase or abiologically active portion thereof, can also be accomplished using atechnology such as real-time Biomolecular Interaction Analysis (BIA).Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 andSzabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705. As used herein,“BIA” is a technology for studying biospecific interactions in realtime, without labeling any of the interactants (e.g., BIAcore). Changesin the optical phenomenon of surface plasmon resonance (SPR) can be usedas an indication of real-time reactions between biological molecules.

In an alternative embodiment, determining the ability of the testcompound to modulate the activity of N-kinase can be accomplished bydetermining the ability of the N-kinase protein to further modulate theactivity of a downstream effector of an N-kinase target molecule. Forexample, the activity of the effector molecule on an appropriate targetcan be determined or the binding of the effector to an appropriatetarget can be determined as previously described.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize any of the reactants, e.g.,N-kinase or a non-N-kinase molecule, to facilitate separation ofcomplexed from uncomplexed forms of one or both of the proteins, as wellas to accommodate automation of the assay. Binding of a test compound toN-kinase, or interaction of N-kinase with a non-N-kinase molecule in thepresence and absence of a test compound, can be accomplished in anyvessel suitable for containing the reactants. Examples of such vesselsinclude microtitre plates, test tubes, and micro-centrifuge tubes. Inone embodiment, a fusion protein can be provided which adds a domainthat allows one or both of the proteins to be bound to a matrix. Forexample, glutathione-S-transferase/N-kinase fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbednon-N-kinase molecule or N-kinase, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level ofN-kinase binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either N-kinaseor a non-N-kinase molecule can be immobilized utilizing conjugation ofbiotin and streptavidin. Biotinylated N-kinase or non-N-kinase moleculecan be prepared from biotin-NHS (N-hydroxy-succinimide) using techniquesknown in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96 wellplates (Pierce Chemical). Alternatively, antibodies reactive withN-kinase or a non-N-kinase molecule but which do not interfere with thebinding of N-kinase to its target non-N-kinase molecule can bederivatized to the wells of the plate, and unbound non-N-kinase moleculeor N-kinase trapped in the wells by antibody conjugation. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with N-kinase or a non-N-kinase molecule, as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with N-kinase or a non-N-kinase target molecule.

In another embodiment, modulators of N-kinase expression are identifiedin a method wherein a cell is contacted with a candidate compound andthe expression of N-kinase mRNA or protein in the cell is determined.The level of expression of N-kinase mRNA or protein in the presence ofthe candidate compound is compared to the level of expression ofN-kinase mRNA or protein in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of N-kinaseexpression based on this comparison. For example, when expression ofN-kinase mRNA or protein is greater (statistically significantlygreater) in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of N-kinase mRNA orprotein expression. Alternatively, when expression of N-kinase mRNA orprotein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound isidentified as an inhibitor of N-kinase mRNA or protein expression. Thelevel of N-kinase mRNA or protein expression in the cells can bedetermined by methods described herein for detecting N-kinase mRNA orprotein.

In yet another aspect of the invention, the N-kinase proteins can beused as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with N-kinase (“N-kinase-binding proteins” or“N-kinase-bp”) and are involved in N-kinase activity. SuchN-kinase-binding proteins are also likely to be involved in thepropagation of signals by the N-kinase proteins or N-kinase targets as,for example, downstream elements of an N-kinase-mediated signalingpathway. Alternatively, such N-kinase-binding proteins are likely to beN-kinase inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for an N-kinaseprotein is fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming anN-kinase-dependent complex, the DNA-binding and activation domains ofthe transcription factor are brought into close proximity. Thisproximity allows transcription of a reporter gene (e.g., LacZ) which isoperably linked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the N-kinase protein.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating compoundcan be identified using a cell-based or a cell free assay, and theability of the compound to modulate the activity of an N-kinase proteincan be confirmed in vivo, e.g., in an animal such as an animal model fora condition characterized by aberrant, e.g., insufficient axonaloutgrowth of central nervous system neurons. Examples of such animalmodels are described in, for example, Benowitz et al. (1999) PNAS96(23): 13486-90. Epilepsy animal models are also known in the art.

This invention further pertains to novel compounds identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use a compound identified as described hereinin an appropriate animal model. For example, a compound identified asdescribed herein (e.g., an N-kinase modulating compound, an antisenseN-kinase nucleic acid molecule, an N-kinase-specific antibody, or anN-kinase-binding partner) can be used in an animal model to determinethe efficacy, toxicity, or side effects of treatment with such acompound. Alternatively, a compound identified as described herein canbe used in an animal model to determine the mechanism of action of sucha compound. Furthermore, this invention pertains to uses of novelcompounds identified by the above-described screening assays fortreatments as described herein.

In one embodiment, the N-kinase used in the methods of the invention isa human N-kinase, such as a recombinantly produced N-kinase. N-kinase,e.g., human N-kinase, may be introduced into a recombinant expressionvector using standard techniques and expressed in prokaryotic oreukaryotic cells. For example, N-kinase, e.g., human N-kinase, can beexpressed in bacterial cells such as E. coli, insect cells (usingbaculovirus expression vectors) yeast cells or mammalian cells. Suitablehost cells are discussed further in Goeddel, Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).Alternatively, a recombinant expression vector containing N-kinase,e.g., human N-kinase, can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

In another embodiment, the N-kinase used in the methods of the inventionis a bovine N-kinase, such as an N-kinase which is purified from abovine source, e.g., neonatal bovine brain tissue, as described hereinin, for instance, Example 1.

In another embodiment, the method of the invention further includesdetermining the ability of the test compound to modulate axonaloutgrowth of a central nervous system neuron. Determining the ability ofthe test compound to modulate axonal outgrowth of a central nervoussystem neuron can be accomplished by, for example, using dissociatedcultures of purified rat retinal ganglion cells. Dissociated cultures ofpurified rat retinal ganglion cells can, for example, be prepared byimmunopanning as described in Barres et al., Neuron, 1: 791-803,1988,the contents of which are incorporated herein by reference. In brief,retinas from Sparague-Dawley rats can be dissociated using papainactivated with cysteine. Macrophages are removed by incubation with ananti-rat macrophage antibody (Accurate) followed by immunopanning withan anti-rabbit IgG antibody. Ganglion cells are isolated byimmunopanning with an anti-Thy-1 antibody, then dislodged with trypsinfor use in low-density cultures. Rat retinal ganglion cells aremaintained at 37° C in a CO₂ incubator using the same medium describedabove except for the presence of 30 mM bicarbonate.

Samples are plated in quadruplicate in randomized positions of a 24-wellplate, contacted with the test compound, and the code is concealed toensure that growth is evaluated in a blinded fashion. Each experimentmay contain 4 wells of a negative control (media plus supplements only)and 4 wells of a positive control (e.g., a standardized AF-1 sample ofknown activity). Growth and survival are assessed after 6 days for allganglion cells in 25 consecutive fields of each well using phasecontrast microscopy at 400× magnification (c. 150 ganglion cells countedper well). Extension of a process 5 cell diameters in length is used asthe criterion for growth, since it clearly distinguishes stimulatedcells from negative controls (Schwalb et al., 1995). After thecompletion of counting, the code is broken, the data tabulated, andmeans and standard errors are calculated for the 4 replicate wells ofeach sample using Cricket Graph (CA Associates, Islandia, N.Y.). Dataare normalized by subtracting the growth in the negative controls(usually 4-5%) and dividing by the net growth in the positive controls.

Goldfish retinal ganglion cells (Benowitz et al. (1998) J. Biol. Chem.273(45):29626-34) as well as mixtures of rat and goldfish ganglion cellsmay also be used.

Compounds that Modulate Axonal Outgrowth of a Central Nervous SystemNeuron

In another aspect, the invention features a compound that modulatesaxonal outgrowth of a central nervous system neuron identified by any ofthe foregoing methods.

In one embodiment, the compound that modulates axonal outgrowth of acentral nervous system neuron is an antisense N-kinase nucleic acidmolecule. An “antisense” nucleic acid comprises a nucleotide sequencewhich is complementary to a “sense” nucleic acid encoding a protein,e.g., complementary to the coding strand of a double-stranded cDNAmolecule or complementary to an mRNA sequence. Accordingly, an antisensenucleic acid can hydrogen bond to a sense nucleic acid. The antisensenucleic acid can be complementary to the entire N-kinase coding strand,or to only a portion thereof. In one embodiment, an antisense nucleicacid molecule is antisense to a “coding region” of the coding strand ofa nucleotide sequence encoding an N-kinase. The term “coding region”refers to the region of the nucleotide sequence comprising codons whichare translated into amino acid residues. In another embodiment, theantisense nucleic acid molecule is antisense to a “noncoding region” ofthe coding strand of a nucleotide sequence encoding N-kinase. The term“noncoding region” refers to 5′ and 3′ sequences which flank the codingregion that are not translated into amino acids (i.e., also referred toas 5′ and 3′ untranslated regions).

Given the coding strand sequence encoding N-kinase, antisense nucleicacids of the invention can be designed according to the rules of Watsonand Crick base pairing. The antisense nucleic acid molecule can becomplementary to the entire coding region of N-kinase mRNA, but morepreferably is an oligonucleotide which is antisense to only a portion ofthe coding or noncoding region of N-kinase mRNA. For example, theantisense oligonucleotide can be complementary to the region surroundingthe translation start site of N-kinase mRNA. An antisenseoligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35,40, 45 or 50 nucleotides in length. An antisense nucleic acid of theinvention can be constructed using chemical synthesis and enzymaticligation reactions using procedures known in the art. For example, anantisense nucleic acid (e.g., an antisense oligonucleotide) can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides which can be used to generate theantisense nucleic acid include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding an N-kinaseprotein to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisensenucleic acid molecules of the invention include direct injection at atissue site, e.g., in the brain. Alternatively, antisense nucleic acidmolecules can be modified to target selected cells and then administeredsystemically. For example, for systemic administration, antisensemolecules can be modified such that they specifically bind to receptorsor antigens expressed on a selected cell surface. e.g., by linking theantisense nucleic acid molecules to peptides or antibodies which bind tocell surface receptors or antigens. The antisense nucleic acid moleculescan also be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

In yet another embodiment, the antisense N-kinase nucleic acid moleculemay be an α-anomeric nucleic acid molecule. An α-anomeric nucleic acidmolecule forms specific double-stranded hybrids with complementary RNAin which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, the compound that modulates axonaloutgrowth of a central nervous system neuron is a ribozyme. Ribozymesare catalytic RNA molecules with ribonuclease activity which are capableof cleaving a single-stranded nucleic acid, such as an mRNA, to whichthey have a complementary region. Thus, ribozymes (e.g., hammerheadribozymes (described in Haselhoff and Gerlach (1988) Nature334:585-591)) can be used to catalytically cleave N-kinase mRNAtranscripts to thereby inhibit translation of N-kinase mRNA. A ribozymehaving specificity for an N-kinase-encoding nucleic acid can be designedbased upon the nucleotide sequence of an N-kinase cDNA. For example, aderivative of a Tetrahymena L-19 IVS RNA can be constructed in which thenucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in an N-kinase-encoding mRNA. See,e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No.5,116,742. Alternatively, N-kinase mRNA can be used to select acatalytic RNA having a specific ribonuclease activity from a pool of RNAmolecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science261:1411-1418.

Alternatively, N-kinase gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of theN-kinase (e.g., the N-kinase promoter and/or enhancers) to form triplehelical structures that prevent transcription of the N-kinase gene intarget cells. See generally, Helene, C. (1991) Anticancer Drug Des.6(6): 569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36;and Maher, L. J. (1992) Bioassays 14(12):807-15.

In still another embodiment, the compound that modulates axonaloutgrowth of a central nervous system neuron is an anti-N-kinaseantibody. A full-length N-kinase protein or, alternatively, antigenicpeptide fragments of N-kinase may be used as immunogens to generateanti-N-kinase antibodies. The antigenic peptide of N-kinase comprises atleast 8 amino acid residues of the amino acid sequence shown in SEQ IDNO:1 and encompasses an epitope of N-kinase such that an antibody raisedagainst the peptide forms a specific immune complex with the N-kinaseprotein. Preferably, the antigenic peptide comprises at least 10 aminoacid residues, more preferably at least 15 amino acid residues, evenmore preferably at least 20 amino acid residues, and most preferably atleast 30 amino acid residues.

Preferred epitopes encompassed by the antigenic peptide are regions ofN-kinase that are located on the surface of the protein, e.g.,hydrophilic regions, as well as regions with high antigenicity.

An N-kinase immunogen typically is used to prepare antibodies byimmunizing a suitable subject, (e.g., rabbit, goat, mouse or othermammal) with the immunogen. An appropriate immunogenic preparation cancontain, for example, recombinantly expressed N-kinase protein or achemically synthesized N-kinase polypeptide. The preparation can furtherinclude an adjuvant, such as Freund's complete or incomplete adjuvant,or similar immunostimulatory agent. Immunization of a suitable subjectwith an immunogenic N-kinase preparation induces a polyclonalanti-N-kinase antibody response.

The term “antibody” as used herein includes immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site which specifically binds(immunoreacts with) an antigen, such as an N-kinase. Examples ofimmunologically active portions of immunoglobulin molecules includeF(ab) and F(ab′)₂ fragments which can be generated by treating theantibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies that bind N-kinase molecules. Theterm “monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope of N-kinase. A monoclonal antibody composition thustypically displays a single binding affinity for a particular N-kinaseprotein with which it immunoreacts.

Polyclonal anti-N-kinase antibodies can be prepared as described aboveby immunizing a suitable subject with an N-kinase immunogen. Theanti-N-kinase antibody titer in the immunized subject can be monitoredover time by standard techniques, such as with an enzyme linkedimmunosorbent assay (ELISA) using immobilized N-kinase. If desired, theantibody molecules directed against N-kinase can be isolated from themammal (e.g., from the blood) and further purified by well knowntechniques, such as protein A chromatography to obtain the IgG fraction.At an appropriate time after immunization, e.g., when the anti-N-kinaseantibody titers are highest, antibody-producing cells can be obtainedfrom the subject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al.(1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem.255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA 76:2927-31;and Yeh et al. (1982) Int. J. Cancer 29:269-75), the more recent human Bcell hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), theEBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. Thetechnology for producing monoclonal antibody hybridomas is well known(see generally R. H. Kenneth, in Monoclonal Antibodies: A New DimensionIn Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980);E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al.(1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line(typically a myeloma) is fused to lymphocytes (typically splenocytes)from a mammal immunized with an N-kinase immunogen as described above,and the culture supernatants of the resulting hybridoma cells arescreened to identify a hybridoma producing a monoclonal antibody thatbinds N-kinase.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-N-kinase monoclonal antibody (see, e.g., G. Galfre et al. (1977)Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra;Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies,cited supra). Moreover, the ordinarily skilled worker will appreciatethat there are many variations of such methods which also would beuseful. Typically, the immortal cell line (e.g., a myeloma cell line) isderived from the same mammalian species as the lymphocytes. For example,murine hybridomas can be made by fusing lymphocytes from a mouseimmunized with an immunogenic preparation of the present invention withan immortalized mouse cell line. Preferred immortal cell lines are mousemyeloma cell lines that are sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a numberof myeloma cell lines can be used as a fusion partner according tostandard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCCTypically, HAT-sensitive mouse myeloma cells are fused to mousesplenocytes using polyethylene glycol (“PEG”). Hybridoma cells resultingfrom the fusion are then selected using HAT medium, which kills unfusedand unproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindN-kinase, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-N-kinase antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with N-kinase to thereby isolateimmunoglobulin library members that bind N-kinase. Kits for generatingand screening phage display libraries are commercially available (e.g.,the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01;and the Stratagene SurZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTInternational Publication No. WO 92/18619; Dower et al. PCTInternational Publication No. WO 91/17271; Winter et al. PCTInternational Publication WO 92/20791; Markland et al. PCT InternationalPublication No. WO 92/15679; Breitling et al. PCT InternationalPublication WO 93/01288; McCafferty et al. PCT International PublicationNo. WO 92/01047; Garrard et al. PCT International Publication No. WO92/09690; Ladner et al. PCT International Publication No. WO 90/02809;Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J.Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gramet al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al.(1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc. AcidRes. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

Additionally, recombinant anti-N-kinase antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.International Application No. PCT/US86/02269; Akira, et al. EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT International Publication No. WO 86/01533; Cabilly et al.U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987)Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol.139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218;Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al. (1985)Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

In one embodiment, the compound that modulates axonal outgrowth of acentral nervous system neuron is an intracellular antibody specific forN-kinase. The use of intracellular antibodies to inhibit proteinfunction in a cell is known in the art (see e.g., Carlson, J. R. (1988)Mol. Cell. Biol. 8:2638-2646; Biocca, S. et al. (1990) EMBO J.9:101-108; Werge, T. M. et al. (1990) FEBS Letters 274:193-198; Carlson,J. R. (1993) Proc. Natl. Acad. Sci. USA 90:7427-7428; Marasco, W. A. etal. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893; Biocca, S. et al.(1994) Bio/Technology 12:396-399; Chen, S-Y. et al. (1994) Human GeneTherapy 5: 595-601; Duan, L et al. (1994) Proc. Natl. Acad. Sci. USA91:5075-5079; Chen, S-Y. et al. (1994) Proc. Natl. Acad. Sci. USA91:5932-5936; Beerli, R. R. et al. (1994) J. Biol. Chem.269:23931-23936; Beerli, R. R. et al. (1994) Biochem. Biophys. Res.Commun. 204:666-672; Mhashilkar, A. M. et al. (1995) EMBO J.14:1542-1551; Richardson, J. H. et al. (1995) Proc. Natl. Acad. Sci. USA92:3137-3141; PCT Publication No. WO 94/02610 by Marasco et al.; and PCTPublication No. WO 95/03832 by Duan et al.).

To inhibit protein activity using an intracellular antibody, arecombinant expression vector is prepared which encodes the antibodychains in a form such that, upon introduction of the vector into a cell,the antibody chains are expressed as a functional antibody in anintracellular compartment of the cell.

In still another embodiment, the compound that modulates axonaloutgrowth of a central nervous system neuron is a small molecule. Asused herein, the term “small molecule” includes, but is not limited to,peptides, peptidomimetics, amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e., including heteroorganicand organometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds.

In still another embodiment, the compound that modulates axonaloutgrowth of a central nervous system neuron is an N-kinase polypeptideor portion thereof, e.g., a fragment that includes at least 15, 20, 25,30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 contiguous amino acids ofthe N-kinase polypeptide. For example, the compound could comprise theconstitutively active catalytic domain of the N-kinase.

In still another embodiment, the compound that modulates axonaloutgrowth of a central nervous system neuron is an N-kinase gene orportion thereof.

Pharmaceutically Acceptable Formulations

Pharmaceutical compositions, and packaged formulations, comprising acomposition of the invention (e.g., compound that modulates the activityof N-kinase) and a pharmaceutically acceptable carrier are also providedby the invention. In the method of the invention, the compound thatmodulates the activity of N-kinase can be administered in apharmaceutically acceptable formulation. Such pharmaceuticallyacceptable formulation typically includes the compound that modulatesthe activity of N-kinase as well as a pharmaceutically acceptablecarrier(s) and/or excipient(s). As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and anti fungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible. Forexample, the carrier can be suitable for injection into thecerebrospinal fluid. Excipients include pharmaceutically acceptablestabilizers and disintegrants. The present invention pertains to anypharmaceutically acceptable formulations, including synthetic or naturalpolymers in the form of macromolecular complexes, nanocapsules,microspheres, or beads, and lipid-based formulations includingoil-in-water emulsions, micelles, mixed micelles, synthetic membranevesicles, and resealed erythrocytes.

In one embodiment, the pharmaceutically acceptable formulations comprisea polymeric matrix. The terms “polymer” or “polymeric” areart-recognized and include a structural framework comprised of repeatingmonomer units which is capable of delivering a compound that modulatesthe activity of N-kinase such that treatment of a targeted condition,e.g., a CNS injury, occurs. The terms also include co-polymers andhomopolymers e.g., synthetic or naturally occurring. Linear polymers,branched polymers, and cross-linked polymers are also meant to beincluded.

For example, polymeric materials suitable for forming thepharmaceutically acceptable formulation employed in the presentinvention, include naturally derived polymers such as albumin, alginate,cellulose derivatives, collagen, fibrin, gelatin, and polysaccharides,as well as synthetic polymers such as polyesters (PLA, PLGA),polyethylene glycol, poloxomers, polyanhydrides, and pluronics. Thesepolymers are biocompatible with the nervous system, including thecentral nervous system, they are biodegradable within the centralnervous system without producing any toxic byproducts of degradation,and they possess the ability to modify the manner and duration ofcompound that modulates the activity of N-kinase release by manipulatingthe polymer's kinetic characteristics. As used herein, the term“biodegradable” means that the polymer will degrade over time by theaction of enzymes, by hydrolytic action and/or by other similarmechanisms in the body of the subject. As used herein, the term“biocompatible” means that the polymer is compatible with a livingtissue or a living organism by not being toxic or injurious and by notcausing an immunological rejection.

Polymers can be prepared using methods known in the art (Sandler, S. R.;Karo, W. Polymer Syntheses; Harcourt Brace: Boston, 1994; Shalaby, W.;Ikada, Y.; Langer, R.; Williams, J. Polymers of Biological andBiomedical Significance (ACS Symposium Series 540; American ChemicalSociety: Washington, D.C., 1994). Polymers can be designed to beflexible; the distance between the bioactive side-chains and the lengthof a linker between the polymer backbone and the group can becontrolled. Other suitable polymers and methods for their preparationare described in U.S. Pat. Nos. 5,455,044 and 5,576,018, the contents ofwhich are incorporated herein by reference.

The polymeric formulations can be formed by dispersion of the compoundthat modulates the activity of N-kinase within liquefied polymer, asdescribed in U.S. Pat. No. 4,883,666, the teachings of which areincorporated herein by reference or by such methods as bulkpolymerization, interfacial polymerization, solution polymerization andring polymerization as described in Odian G., Principles ofPolymerization and ring opening polymerization, 2nd ed., John Wiley &Sons, New York, 1981, the contents of which are incorporated herein byreference. The properties and characteristics of the formulations arecontrolled by varying such parameters as the reaction temperature,concentrations of polymer and compound that modulates the activity ofN-kinase, types of solvent used, and reaction times.

The compound that modulates the activity of N-kinase can be encapsulatedin one or more pharmaceutically acceptable polymers, to form amicrocapsule, microsphere, or microparticle, terms used hereininterchangeably. Microcapsules, microspheres, and microparticles areconventionally free-flowing powders consisting of spherical particles of2 millimeters or less in diameter, usually 500 microns or less indiameter. Particles less than 1 micron are conventionally referred to asnanocapsules, nanoparticles or nanospheres. For the most part, thedifference between a microcapsule and a nanocapsule, a microsphere and ananosphere, or microparticle and nanoparticle is size; generally thereis little, if any, difference between the internal structure of the two.In one aspect of the present invention, the mean average diameter isless than about 45 μm, preferably less than 20 μm, and more preferablybetween about 0.1 and 10 μm.

In another embodiment, the pharmaceutically acceptable formulationscomprise lipid-based formulations. Any of the known lipid-based drugdelivery systems can be used in the practice of the invention. Forinstance, multivesicular liposomes (MVL), multilamellar liposomes (alsoknown as multilamellar vesicles or “MLV”), unilamellar liposomes,including small unilamellar liposomes (also known as unilamellarvesicles or “SUV”) and large unilamellar liposomes (also known as largeunilamellar vesicles or “LUV”), can all be used so long as a sustainedrelease rate of the encapsulated compound that modulates the activity ofN-kinase or analogue thereof can be established. In one embodiment, thelipid-based formulation can be a multivesicular liposome system. Methodsof making controlled release multivesicular liposome drug deliverysystems is described in PCT Application Serial Nos. US96/11642,US94/12957 and US94/04490, the contents of which are incorporated hereinby reference.

The composition of the synthetic membrane vesicle is usually acombination of phospholipids, usually in combination with steroids,especially cholesterol. Other phospholipids or other lipids may also beused.

Examples of lipids useful in synthetic membrane vesicle productioninclude phosphatidylglycerols, phosphatidylcholines,phosphatidylserines, phosphatidylethanolamines, sphingolipids,cerebrosides, and gangliosides. Preferably phospholipids including eggphosphatidylcholine, dipalmitoylphosphatidylcholine,distearoylphosphatidylcholine, dioleoylphosphatidylcholine,dipalmitoylphosphatidylglycerol, and dioleoylphosphatidylglycerol areused.

In preparing lipid-based vesicles containing a compound that modulatesthe activity of N-kinase or analogue thereof, such variables as theefficiency of compound that modulates the activity of N-kinaseencapsulation, lability of the compound that modulates the activity ofN-kinase, homogeneity and size of the resulting population of vesicles,compound that modulates the activity of N-kinase-to-lipid ratio,permeability, instability of the preparation, and pharmaceuticalacceptability of the formulation should be considered (see Szoka, etal., Annual Reviews of Biophysics and Bioengineering, 9:467, 1980;Deamer, et al., in Liposomes, Marcel Dekker, New York, 1983, 27; andHope, et al., Chem. Phys. Lipids, 40:89, 1986, the contents of which areincorporated herein by reference).

Administration of the Pharmaceutically Acceptable Formulation

The pharmaceutically acceptable formulations of the invention areadministered such that the compound that modulates the activity ofN-kinase, or analogue thereof, comes into contact with central nervoussystem neurons to thereby modulate the axonal outgrowth thereof. Bothlocal and systemic administration of the formulations are contemplatedby the invention, although local administration may be preferable toachieve effective local concentrations of the compound that modulatesthe activity of N-kinase, or analogue, as well as to avoid possible sideeffects from systemic administration of the agent. In one embodiment,the compound that modulates the activity of N-kinase is administered byintroduction into the central nervous system of the subject, e.g., intothe cerebrospinal fluid of the subject. In certain aspects of theinvention, the compound that modulates the activity of N-kinase isintroduced intrathecally, e.g., into a cerebral ventricle, the lumbararea, or the cisterna magna. In another aspect, the compound thatmodulates the activity of N-kinase is introduced intraocularly, tothereby contact retinal ganglion cells.

The pharmaceutically acceptable formulations can easily be suspended inaqueous vehicles and introduced through conventional hypodermic needlesor using infusion pumps. Prior to introduction, the formulations can besterilized with, preferably, gamma radiation or electron beamsterilization, described in U.S. Pat. No. 436,742 the contents of whichare incorporated herein by reference.

In one embodiment, the compound that modulates the activity of N-kinaseformulation described herein is administered to the subject in theperiod from the time of, for example, an injury to the CNS up to about100 or 200 hours after the injury has occurred, for example, within 48,36, 24, 12, or 6 hours from the time of injury. In another embodiment,the compound that modulates the activity of N-kinase is administered toa subject suffering from a chronic injury to the CNS. Accordingly, thecompound is administered to the subject over the subject's life time.

In another embodiment of the invention, the compound that modulates theactivity of N-kinase formulation is administered into a subjectintrathecally. As used herein, the term “intrathecal administration” isintended to include delivering a compound that modulates the activity ofN-kinase formulation directly into the cerebrospinal fluid of a subject,by techniques including lateral cerebroventricular injection through aburrhole or cisternal or lumbar puncture or the like (described inLazorthes et al. Advances in Drug Delivery Systems and Applications inNeurosurgery, 143-192 and Omaya et al., Cancer Drug Delivery, 1:169-179, the contents of which are incorporated herein by reference).The term “lumbar region” is intended to include the area between thethird and fourth lumbar (lower back) vertebrae. The term “cisternamagna” is intended to include the area where the skull ends and thespinal cord begins at the back of the head. The term “cerebralventricle” is intended to include the cavities in the brain that arecontinuous with the central canal of the spinal cord. Administration ofa compound that modulates the activity of N-kinase to any of the abovementioned sites can be achieved by direct injection of the compound thatmodulates the activity of N-kinase formulation or by the use of infusionpumps. For injection, the compound that modulates the activity ofN-kinase formulation of the invention can be formulated in liquidsolutions, preferably in physiologically compatible buffers such asHank's solution or Ringer's solution. In addition, the compound thatmodulates the activity of N-kinase formulation may be formulated insolid form and re-dissolved or suspended immediately prior to use.Lyophilized forms are also included. The injection can be, for example,in the form of a bolus injection or continuous infusion (e.g., usinginfusion pumps) of the compound that modulates the activity of N-kinaseformulation.

In one embodiment of the invention, the compound that modulates theactivity of N-kinase formulation is administered by lateral cerebroventricular injection into the brain of a subject, preferably within 100hours of when an injury (resulting in a condition characterized byaberrant axonal outgrowth of central nervous system neurons) occurs(e.g., within 6, 12, or 24 hours of the time of the injury). Theinjection can be made, for example, through a burr hole made in thesubject's skull. In another embodiment, the formulation is administeredthrough a surgically inserted shunt into the cerebral ventricle of asubject, preferably within 100 hours of when an injury occurs (e.g.,within 6, 12 or 24 hours of the time of the injury). For example, theinjection can be made into the lateral ventricles, which are larger,even though injection into the third and fourth smaller ventricles canalso be made. In yet another embodiment, the compound that modulates theactivity of N-kinase formulation is administered by injection into thecisterna magna, or lumbar area of a subject, preferably within 100 hoursof when an injury occurs (e.g., within 6, 12, or 24 hours of the time ofthe injury).

In another embodiment of the invention, the compound that modulates theactivity of N-kinase formulation is administered to a subject at thesite of injury, preferably within 100 hours of when an injury occurs(e.g., within 6, 12, or 24 hours of the time of the injury).

Duration and Levels of Administration

In a preferred embodiment of the method of the invention, the compoundthat modulates the activity of N-kinase, or analog thereof, is contactedwith CNS neurons for an extended period of time to effect modulation ofaxonal outgrowth. Sustained contact with the compound that modulates theactivity of N-kinase, or analog, can be achieved by, for example,repeated administration of the compound that modulates the activity ofN-kinase or analog over a period of time, such as one week, severalweeks, one month or longer. More preferably, the pharmaceuticallyacceptable formulation used to administer the compound that modulatesthe activity of N-kinase, or analog, provides sustained delivery, e.g.,“slow release” of the compound that modulates the activity of N-kinase,or analog, to a subject. For example, the formulation may deliver thecompound that modulates the activity of N-kinase, or analog, for atleast one, two, three, or four weeks after the pharmaceuticallyacceptable formulation is administered to the subject. Preferably, asubject to be treated in accordance with the present invention istreated with the compound that modulates the activity of N-kinase, oranalog, for at least 30 days (either by repeated administration or byuse of a sustained delivery system, or both).

As used herein, the term “sustained delivery” is intended to includecontinual delivery of a compound that modulates the activity of N-kinaseor analogue thereof in vivo over a period of time followingadministration, preferably at least several days, a week, several weeks,one month or longer. Sustained delivery of the compound that modulatesthe activity of N-kinase or analogue thereof can be demonstrated by, forexample, the continued therapeutic effect of the compound that modulatesthe activity of N-kinase or analogue thereof over time (e.g., sustaineddelivery of the compound that modulates the activity of N-kinase oranalogue thereof can be demonstrated by continued outgrowth or bycontinued inhibition of outgrowth of CNS neurons over time).Alternatively, sustained delivery of the compound that modulates theactivity of N-kinase or analogue thereof may be demonstrated bydetecting the presence of the compound that modulates the activity ofN-kinase or analogue thereof in vivo over time.

Preferred approaches for sustained delivery include use of a polymericcapsule or a minipump to deliver the formulation. Polymeric capsules canbe prepared as described herein. Implantable infusion pump systems(e.g., Infusaid; see e.g., Zierski, J. et al. (1988) Acta Neurochem.Suppl. 43:94-99; Kanoff, R. B. (1994) J. Am. Osteopath. Assoc.94:487-493) and osmotic pumps (sold by Alza Corporation) are availablein the art. Another mode of administration is via an implantable,externally programmable infusion pump. Suitable infusion pump systemsand reservoir systems are also described in U.S. Pat. No. 5, 368,562 byBlomquist and U.S. Pat. No. 4,731,058 by Doan, developed by PharmaciaDeltec Inc.

The pharmaceutical formulation, used in the method of the invention,contains a therapeutically effective amount of the compound thatmodulates the activity of N-kinase or analogue thereof. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredresult. A therapeutically effective amount of the compound thatmodulates the activity of N-kinase or analogue thereof may varyaccording to factors such as the disease state, age, and weight of thesubject, and the ability of the compound that modulates the activity ofN-kinase or analogue thereof (alone or in combination with one or moreother agents) to elicit a desired response in the subject. Dosageregimens may be adjusted to provide the optimum therapeutic response. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the compound that modulates the activity ofN-kinase or analogue thereof are outweighed by the therapeuticallybeneficial effects. A non-limiting dosage range is about 5 μM-1000 μM,although the particular optimal dosage will vary depending upon, amongother factors, the particular compound that modulates the activity ofN-kinase, or analogue thereof, used.

It is to be noted that dosage values may vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompound that modulates the activity of N-kinase or analogue thereof andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed invention.

The invention, in another embodiment, provides a pharmaceuticalcomposition consisting essentially of a compound that modulates theactivity of N-kinase and a pharmaceutically acceptable carrier, as wellas methods of use thereof to modulate axonal outgrowth by contacting CNSneurons with the composition. By the term “consisting essentially of” ismeant that the pharmaceutical composition does not contain any othermodulators of neuronal growth such as, for example, nerve growth factor(NGF). In one embodiment, the pharmaceutical composition of theinvention can be provided as a packaged formulation. The packagedformulation may include a pharmaceutical composition of the invention ina container and printed instructions for administration of thecomposition for treating a subject having a disorder associated with aninjury of central nervous system neurons, e.g., an injury to retinalganglion cells, a spinal cord injury or a traumatic brain injury. Use ofthe compound that modulates the activity of N-kinases, and analoguesthereof, of the invention in the manufacture of a medicament formodulating the outgrowth of CNS neurons (e.g., mammalian CNS neurons) isalso encompassed by the invention.

In Vitro Treatment of CNS Neurons

CNS neurons can further be contacted with a compound that modulates theactivity of N-kinase, in vitro to modulate axonal outgrowth in vitro.Accordingly, CNS neuron cells can be isolated from a subject and grownin vitro, using techniques well known in the art, and then treated inaccordance with the present invention to modulate axonal outgrowth.Briefly, a CNS neuron cell culture can be obtained by allowing neuroncells to migrate out of fragments of neural tissue adhering to asuitable substrate (e.g., a culture dish) or by disaggregating thetissue, e.g., mechanically or enzymatically, to produce a suspension ofCNS neuron cells. For example, the enzymes trypsin, collagenase,elastase, hyaluronidase, DNase, pronase, dispase, or variouscombinations thereof can be used. Trypsin and pronase give the mostcomplete disaggregation but may damage the cells. Collagenase anddispase give a less complete disaggregation but are less harmful.Methods for isolating tissue (e.g., neural tissue) and thedisaggregation of tissue to obtain cells (e.g., CNS neuron cells) aredescribed in Freshney R. I., Culture of Animal Cells, A Manual of BasicTechnique, Third Edition, 1994, the contents of which are incorporatedherein by reference.

Such cells can be subsequently contacted with a compound that modulatesthe activity of N-kinase in amounts and for a duration of time asdescribed above. Once modulation of axonal outgrowth has been achievedin the CNS neuron cells, these cells can be re-administered to thesubject, e.g., by implantation. It is preferred that the cells are notallowed to differentiate extensively in vitro, as cells that integratemost successfully in a subject are primitive cells.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures and the Sequence Listing arehereby incorporated by reference.

EXAMPLES Example I Isolation and Characterization of the N-kinasePolypeptide

Neocortical gray matter from bovine brain was homogenized in buffercontaining protease and phosphatase inhibitors, and particulate materialwas centrifuged down. The soluble fraction from approximately 1 kg oftissue was used as the starting material for isolation of the kinase.

During the purification process, kinase activity was monitored using anin-gel kinase method. In this method, a histone HF-1 substrate proteinis polymerized into a 10% polyacrylamide gel before the samples to beanalyzed are electrophoresed in the gel. Following completion of theelectrophoresis, proteins are partially renatured with guanidiumisothiocyanate and incubated in the presence [³²P]-ATP plus Mn²⁺, withor without 6-TG present. (Activation by Mn²⁺, but not by Mg²⁺ or Ca²⁺,is a distinctive property of N-kinase). The purification of the kinasewas monitored by looking for a 6-TG-inhibitable radioactive bandcorresponding to the site where the kinase phosphorylates the HF-1substrate in the gel.

In the first step of the purification process the starting material wassubjected to cation-exchange chromatography (using a Fast-S column,Pharmacia). A 6-TG inhibitable kinase activity bound strongly to thiscolumn and eluted with 0.3 M NaCl. The cation-exchange column fractioncontaining N-kinase was subsequently separated on a Cibacron Blue column(Pharmacia), which allows for the separation of adeninenucleotide-binding proteins. A 6-TG inhibitable, 47-50 kDa polypeptidebound strongly and required a NaCl concentration of 1.5 M NaCl forelution (see FIG. 1A).

The eluted fraction containing the N-kinase polypeptide was, then,subjected to reversed-phase chromatography with a C4 hydrophobicinteraction column (Pharmacia). Briefly, the Cibacron Blue columnfraction containing the N-kinase was applied to the C4 column and elutedwith a gradient of increasing acetonitrile-isopropanol concentration.Evaluation of the kinase activity of the column fractions by in-gelkinase assays showed that the 6-TG-inhibitable, HF-1-phosphorylatingactivity eluted in fractions 24-26 (FIG. 1B). To achieve a higher levelof purification, these fractions were pooled, re-applied to the samecolumn, and the separation was repeated with a gradient of increasingacetonitrile-isopropanol concentration. The N-kinase polypeptide, again,eluted at fractions 24-26.

The column fractions containing the highest concentrations of N-kinasewere lyophilized and applied to a 10% polyacrylamide SDS gel. A smallportion of the sample was run on a parallel gel to carry out in-gelkinase assays. A band at 49 kDa was clearly visible after staining thegel with Coomassie blue; this coincided in its migration position withthe 6-TG-inhibitable kinase activity. This band was cut out and it wasverified that it contained HF-1-phosphorylating activity.

The gel band containing the N-kinase polypeptide was then subjected topartial proteolytic digestion, the proteolytic fragments were analyzedby mass spectroscopy and the masses of the fragments compared to thoseof various known peptides using the process described in, for example,Eng J. K. et al. (1994) J. Am. Soc. Mass. Spectrom. 5:976-989; ChittumH. S. et al. (1998) Biochemistry 37:10866-870; and LeRoy G. et al.(1998) Science 282:1900-04, the contents of which are incorporatedherein by reference.

This analysis revealed that N-kinase is an isoform of MST-3, i.e.,either MST-3 itself, MST-3b, or an as yet undefined isoform. Theseproteins are members of the STE family of serine-threonine kinases thatare found throughout the animal kingdom. STE family members aregenerally components of modular signaling cassettes that are involved invarious aspects of cellular differentiation. FIG. 2 depicts the aminoacid sequence of the N-kinase. Direct matches between the purifiedprotein and the published sequence are shown in blue. K65 (bold type)lies in the ATP-binding region of the kinase domain.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for treating a subject suffering or prone to suffering froma condition characterized by aberrant axonal outgrowth of centralnervous system neurons, comprising administering to said subject acompound that modulates the activity of N-kinase, thereby treating thesubject suffering or prone to suffering from a condition characterizedby aberrant axonal outgrowth of central nervous system neurons.
 2. Themethod of claim 1, wherein the condition characterized by aberrantaxonal outgrowth of central nervous system neurons is spinal cordinjury.
 3. The method of claim 2, wherein the spinal cord injury isselected from the group consisting of monoplegia, diplegia, paraplegia,hemiplegia and quadriplegia.
 4. The method of claim 1, wherein thecondition characterized by aberrant axonal outgrowth of central nervoussystem neurons is epilepsy.
 5. The method of claim 4, wherein theepilepsy is posttraumatic epilepsy.
 6. The method of claim 1, whereinthe condition characterized by aberrant axonal outgrowth of centralnervous system neurons is neuropathic pain syndrome.
 7. The method ofclaim 1, wherein the compound that modulates the activity of N-kinase isadministered by introduction into the central nervous system of thesubject.
 8. The method of claim 1, wherein the compound that modulatesthe activity of N-kinase is introduced into the cerebrospinal fluid ofthe subject.
 9. The method of claim 1, wherein the compound thatmodulates the activity of N-kinase is introduced to the subjectintrathecally.
 10. The method of claim 1, wherein the compound thatmodulates the activity of N-kinase is introduced into a cerebralventricle of the subject.
 11. The method of claim 1, wherein thecompound that modulates the activity of N-kinase is introduced into thelumbar area of the subject.
 12. The method of claim 1, wherein thecompound that modulates the activity of N-kinase is introduced into thecisterna magna of the subject.
 13. The method of claim 1, wherein thecompound that modulates the activity of N-kinase is administered to thesubject in a pharmaceutically acceptable formulation.
 14. The method ofclaim 13, wherein the pharmaceutically acceptable formulation is adispersion system.
 15. The method of claim 13, wherein thepharmaceutically acceptable formulation comprises a lipid-basedformulation.
 16. The method of claim 15, wherein the pharmaceuticallyacceptable formulation comprises a liposome formulation.
 17. The methodof claim 16 wherein the pharmaceutically acceptable formulationcomprises a multivesicular liposome formulation.
 18. The method of claim13, wherein the pharmaceutically acceptable formulation comprises apolymeric matrix.
 19. The method of claim 13, wherein thepharmaceutically acceptable formulation is contained within a minipump.20. The method of claim 13, wherein the pharmaceutically acceptableformulation provides sustained delivery of the compound that modulatesthe activity of N-kinase, to a subject for at least one week after thepharmaceutically acceptable formulation is administered to the subject.21. The method of claim 13, wherein the pharmaceutically acceptableformulation provides sustained delivery of the compound that modulatesthe activity of N-kinase, to a subject for at least one month after thepharmaceutically acceptable formulation is administered to the subject.22. The method of claim 1, wherein the subject is a mammal.
 23. Themethod of claim 22, wherein the mammal is a human.
 24. The method ofclaim 1, wherein the central nervous system neurons are retinal ganglioncells.
 25. A method for modulating axonal outgrowth of a central nervoussystem neuron, comprising contacting the central nervous system neuronwith a compound that modulates the activity of N-kinase, therebymodulating axonal outgrowth of the central nervous system neuron. 26.The method of claim 25, wherein the outgrowth is stimulated.
 27. Themethod of claim 25, wherein the outgrowth is inhibited.
 28. The methodof claim 25, wherein said central nervous system neurons are mammalian.29. A method for modulating the axonal outgrowth of a central nervoussystem neuron in a subject, comprising administering to said subject acompound that modulates the activity of N-kinase, such that axonaloutgrowth in the subject is modulated.
 30. A method for identifying acompound that modulates axonal outgrowth of a central nervous systemneuron, comprising contacting N-kinase with a test compound anddetermining the ability of the test compound to modulate the activity ofN-kinase, thereby identifying a compound that modulates axonal outgrowthof a central nervous system neuron.
 31. The method of claim 30, whereinthe N-kinase is human N-kinase.
 32. The method of claim 31, wherein thehuman N-kinase is a recombinantly produced N-kinase.
 33. The method ofclaim 30, wherein the N-kinase is bovine N-kinase.
 34. The method ofclaim 33, wherein the bovine N-kinase is purified from a bovine source.35. The method of claim 30, further comprising determining the abilityof the test compound to modulate axonal outgrowth of a central nervoussystem neuron.
 36. The method of claim 30, wherein the test compoundinhibits the activity.
 37. The method of claim 30, wherein the testcompound stimulates the activity.
 38. The method of claim 30, whereinthe ability of the test compound to modulate the activity of N-kinase isdetermined by assessing the ability of the test compound to modulateN-kinase dependent phosphorylation of a substrate.
 39. A method foridentifying a compound that modulates axonal outgrowth of a centralnervous system neuron, comprising contacting N-kinase with a testcompound, an N-kinase substrate, radioactive ATP, and Mn⁺²; anddetermining the ability of the test compound to modulate N-kinasedependent phosphorylation of the substrate, thereby identifying acompound that modulates axonal outgrowth of a central nervous systemneuron.
 40. The method of claim 39, wherein the N-kinase substrate is ahistone HF-1 protein.
 41. The method of claim 39, wherein theradioactive ATP is [γ-³²P] ATP.
 42. The method of claim 39, wherein theN-kinase is human N-kinase.
 43. The method of claim 42, wherein thehuman N-kinase is a recombinantly produced N-kinase.
 44. The method ofclaim 39, wherein the N-kinase is bovine N-kinase.
 45. The method ofclaim 44, wherein the bovine N-kinase is purified from a bovine source.46. The method of claim 39, further comprising determining the abilityof the test compound to modulate axonal outgrowth of a central nervoussystem neuron.
 47. A compound that modulates axonal outgrowth of acentral nervous system neuron identified by the method of claim
 30. 48.A compound that modulates axonal outgrowth of a central nervous systemneuron identified by the method of claim
 39. 49. An isolated N-kinasepolypeptide of the type that: (a) is present in neonatal brain tissue;(b) is inhibited in the presence of 6-thioguanine; (c) is activated byMn⁺² but not by Mg⁺² or Ca⁺²; (d) has a molecular weight ofapproximately 49 kDa; and (e) is eluted from a Cibacron Blue column at aNaCl concentration of 1.5-1.75 M.
 50. An antibody which is specificallyreactive with an epitope of the N-kinase polypeptide of claim
 49. 51.The antibody of claim 50, wherein the antibody is an intracellularantibody.
 52. The antibody of claim 50, wherein the epitope comprises anATP binding domain.
 53. A fragment of the N-kinase polypeptide of claim49, wherein the fragment comprises at least 15 contiguous amino acids.54. The fragment of claim 53, wherein the fragment comprises at least 30contiguous amino acids.
 55. The fragment of claim 53, wherein thefragment comprises at least 50 contiguous amino acids.
 56. The fragmentof claim 53, wherein the fragment comprises at least 100 contiguousamino acids.
 57. A fragment of the N-kinase polypeptide of claim 49,wherein the fragment is able to elicit an immune response.